Enzymes responsible for the metabolism of cis-zeatin

ABSTRACT

Isolated nucleic acids encoding cis-zeatin O-glucosyltransferase are disclosed. These nucleic acid molecules are useful, among other things, to produce transgenic plants having modified cis-zeatin O-glucosyltransferase activity and/or modified growth and developmental patterns.

CROSS REFERENCE TO RELATED APPLICATIONS

This is the U.S. National Stage of International Application No.PCT/US01/18406, filed Jun. 5, 2001, which was published in English underPCT Article 21(2), which in turn claims the benefit of U.S. ProvisionalApplication No. 60/209,842, filed Jun. 6, 2000. Both applications areincorporated herein in their entirety.

FIELD OF THE DISCLOSURE

This disclosure relates to plant hormones and in particular tocytokinins. Aspects of the disclosure include a purified cis-zeatinO-glucosyltransferase enzyme, nucleic acid molecules encoding thisenzyme, and vectors containing all or a portion of the nucleic acidmolecule. Transgenic cells and transgenic plants having modifiedcis-zeatin O-glucosyltransferase activity are also provided. Thedisclosure also relates to altered plant traits in general, and seeddevelopment and yield in particular, resulting from the modification ofcis-zeatin O-glucosyltransferase activity in plants.

BACKGROUND

Cytokinins are plant hormones that mediate cell division anddevelopment. This group of hormones was discovered by Miller et al.(Miller et al., Journal of American Chemical Society 77:1392, 1955;Miller et al., Journal of American Chemical Society 78:1375–1380, 1956)with the identification of the first synthetic cytokinin, kinetin. Thefirst naturally occurring cytokinin, zeatin (trans-zeatin), wasdiscovered by Letham (Letham et al., Ann. Botany 41:261–263, 1976) incorn, and the structure of zeatin was determined by Shaw and Wilson(Shaw et al., Proceedings of Chemical Society 231, 1964). Zeatin is themost active and ubiquitous cytokinin in all plant species examined todate. Other naturally occurring cytokinins are structurally related tozeatin (Shaw, Cytokinins, Chemistry, Activity and Function, Mok and Mok,CRC Press, 15–34, 1994).

The critical importance of cytokinins in plant development wasillustrated by the classic tissue culture experiments of Skoog et al.(Skoog et al., Science 148:532, 1965). These experiments establishedthat plant cell division requires cytokinin. Furthermore, the ratio ofcytokinins to auxins (another group of plant hormones) was shown toindicate whether undifferentiated plant cells would develop into shoots(high cytokinin to auxin) or roots (low cytokinin to auxin), or continueto proliferate as callus tissues (intermediate cytokinin to auxinratio). Thereafter, cytokinin was found to be involved in every phase ofplant growth (Mok, Cytokinins, Chemistry, Activity and Function, Mok andMok, CRC Press, 155–166, 1994). In general, cytokinins have growthpromoting effects, from seed germination and shoot development toretarding senescence and increasing fruit and seed set

The effects of cytokinins in controlling plant growth have beenextensively utilized in plant tissue culture to micropropagate and cloneplants and to regenerate whole plants from cells of many species(Krikorian, Plant Hormones-Physiology, Biochemistry and MolecularBiology, 2^(nd) Edition, Davies, Kluwer Academic Publishers, 774–796,1995). In fact, the application of cytokinins in vitro contributessignificantly to advances in plant biotechnology. In agriculturalapplications, external applications of cytokinins on whole plants areused to obtain enhanced fruit set and gram yield of food crops andlonger shelf life of ornamentals (Hradecka et al., Physiology andBiochemistry of Cytokinins in Plants, Kaminek et al., SPB AcademicPublishing, 245–247, 1992; Karanov et al., Progress in Plant GrowthRegulation, Karssen et al., 842–851, 1992; Lewis et al., Physiol. Plant.98:187–195, 1996; Minana et al., J. Exp. Bot. 219:1127–1134, 1989).

In whole plants, cytokinins are synthesized in the roots and transportedto above ground parts (Letham, Cytokinins: Chemistry, Activity andFunction, Mok, and Mok, CRC Press, Boca Raton, 57–80, 1994), althoughother actively growing tissues also have biosynthetic capacity. Twobiosynthetic pathways have been proposed for cytokinin biosynthesis. Thefirst is the direct pathway, involving formation ofN⁶-isopentenyladenosine phosphate from AMP and dimethylallylpyrophosphate, followed by hydroxylation of the side chain to formzeatin-type compounds. The second pathway is the indirect pathway, inwhich cytokinins are released by turnover of tRNA containing cis-zeatin(Prinsen et al., Plant Growth Regul. 23:3–15, 1997). Plant AMPisopentenyltransferases have not been found in spite of theidentification of such genes from bacteria such as Agrobacteriumtumefaciens (Akiyoshi et al., Proc. Natl. Acad. Sci. USA 81:5994–5998,1984; Barry et al., Proc. Natl. Acad. Sci. 81:4776–4780, 1984; Beaty etal., Mol. Gen. Genet. 203:274–280, 1986). In fact, plant DNA homologousto these bacterial genes has not been reported. Therefore, theintermediates, the enzymes, and the genes involved in direct pathway(s)of cytokinin biosynthesis in plants remain unproven or unknown.Cytokinins occur adjacent to the anticodon in tRNAs recognizing codonsbeginning with U (Skoog et al., Ann. Rev. Plant Physiol. 21:359–384,1970; Taller, Cytokinins, Chemistiy, Activity and Function, Mok and Mok,CRC Press, 101–112, 1994). The indirect pathway involves release ofcytokinins from breakdown of such tRNA. Although the weakly activecis-zeatin is the major cytokinin in plant tRNA, cis-zeatin can beconverted to trans-zeatin by cis-trans isomerization (Bassil et al.,Plant Physiol. 102:867–872, 1993).

Although trans-zeatin and its derivatives are prevalent in most plants,cis-zeatin has been found in potato (Mauk et al., Plant Physiol.62:438–442, 1978), tobacco (Tay et al., Plant Sciences 43:131–134,1986), rice (Izumi et al., Plant Cell Physiol. 29:97–104, 1988), and asthe predominant cytokinin in chickpeas (Emery et al., Plant Physiol.117:1515–1523, 1998). Relatively high levels of cis-zeatin occurs inbelow-ground parts of the plants such as roots and tubers. Cones of hops(Watanabe et al., Plant and Cell Physiol. 22:489–500, 1981) and unisexflowers of Mercurialis (Durand et al., Cytokinins, Chemistry, Activityand Function, Mok and Mok, CRC Press, 295–304, 1994) also contain muchcis-zeatin. Therefore, cis-zeatin may play a unique role in biosynthesisas well as in mediating specific developmental steps not yet discovered.

Cytokinins are converted to various metabolites in plant tissues(Jameson, Cytokinins, Chemistry, Activity and Function, Mok and Mok, CRCPress, 113–128, 1994). For example, the metabolites of zeatin includeO-glycosylzeatin, N-glucosylzeatin, zeatin riboside, and zeatinnucleotides. The precise functions of these metabolites are stilluncertain. However, some may be the stored or the transported form ofthe active compound, zeatin. O-Glucoside of zeatin (FIG. 1) may be sucha metabolite (Badenoch-Jones et al., Plant Cell and Environment19:504–516, 1996). Trans-Zeatin O-glucoside was first discovered byLetham et al. (Letham et al., Ann. Botany 41:261–263, 1976) and has beenfound in all crops examined including corn, beans, poplar, soybean, etc.As O-glucosylzeatin can be readily converted back to its active form,zeatin, by the removal of the glucose moiety (via the action: ofwide-spread enzymes, β-glucosidases), O-glucosylzeatin is considered areversible reserve of active cytoknin (Brzobohaty et al., Science262:1051–1054, 1993). Also, O-glucosylzeatin is resistant to attack bycytokinin oxidases (McGaw et al., Planta 159:30–37, 1983) that degradethe parent compound, zeatin. Therefore, O-glucosylzeatin may beimportant in cytokinin action by serving as an interchangeable reserveand as an oxidase resistant form of zeatin. Another metabolite, zeatinO-xyloside was fist discovered in beans (Phaseolus) by Lee et al., (Leeet al., Plant Physiol. 77:635–641, 1985). Zeatin O-xyloside is alsoresistant to degradation and can be reconverted to zeatin. Two enzymes,zeatin O-glucosyltransferase (ZOG) and zeatin O-xylosyltransferase(ZOX), catalyzing the formation of zeatin to O-glucosylzeatin andO-xylosylzeatin, respectively, were first purified and characterized inthe Mok laboratory (Turner et al., Proc. Natl. Acad. Sci. 84:3714–3717,1987; Dixon et al., Plant Physiology 90:1316–1321, 1989). The occurrenceof the enzymes is species specific. The former was isolated from limabeans (Phaseolus lunatus) and the latter from common beans (P.vulgaris). The isolation of the enzymes was followed by the generationof specific antibodies recognizing the enzymes (Martin et al., PlantPhysiology 94:1290–1294, 1990). Subsequently, two genes encoding therespective enzymes were cloned (Martin et al., Proc. Natl. Acad Sci. USA96:284–289, 1999; Martin et al., Plant Physiol. 120:553–557, 1999). Thegenes were designated as ZOG1 (for zeatin O-glucosyltransferase) andZOX1 (for zeatin O-xylosyltransferase).

Plants having modified endogenous zeatin activity would be ofsignificant agricultural importance. Such plants could be createdthrough genetic engineering if the genes regulating zeatin wereavailable. It is to such genes, and polypeptides encoded thereby, thatthe present disclosure is directed.

SUMMARY OF THE DISCLOSURE

The present disclosure provides isolated plant nucleic acid molecules(cDNA and ORF sequences) encoding cis-zeatin O-glucosyltransferase(cis-ZOG1), a key enzyme in the regulation of zeatin activity in plants.

In one embodiment, the cis-ZOG1 nucleic acids disclosed are from corn,Zea mays. The open reading frame of these nucleic acid molecules encodesa polypeptide of 467 amino acids in length. This polypeptide is shown tohave cis-ZOG1 enzymatic activity i.e., the enzyme catalyzes theconversion of cis-zeatin to cis-O-glucosylzeatin. Accordingly, oneaspect comprises isolated nucleic acid molecules encoding cis-ZOG1.Another aspect is the purified cis-ZOG1 enzyme, and fragments andvariants thereof that maintain substantial (e.g., greater than 50% ofthe native protein) catalytic activity.

Also encompassed within the scope of this disclosure are transformationvectors that include at least a portion of the disclosed nucleic acidsequences. Such vectors may be transformed into plants to producetransgenic plants having modified cis-ZOG1 activity. Depending on theparticular sequence incorporated into the vector, transformation withthe cis-ZOG1 cDNA, genes, or derivatives thereof may be used to modifyagronomically important traits, including the activity of zeatin inseeds, grain yield, seed germination rates, and plant growth. While allcrop plants may benefit from such modified activity, it is believed thatthe disclosure will be particularly valuable in Zea mays, wheat, rice,potato, and legumes.

Typically, vectors used to modify cis-ZOG1 activity include regulatorysequences that are operably linked to the cis-ZOG1 cDNA, ORF, orderivatives thereof. For example, cis-ZOG1 activity may be modified inplants by introducing a transformation vector that includes a sense orantisense form of the disclosed cDNA operably linked to a high-levelconstitutive promoter such as the 35S promoter of cauliflower mosaicvirus. Transgenic plants tansformed with such recombinant vectors andhaving modified cis-ZOG1 activity are part of the disclosure.

The disclosure provides cis-ZOG1-encoding nucleic acids from Zea mays,and it additionally encompasses homologs, orthologs and derivatives ofthese sequences, as well as homologs, orthologs, and variants of thecis-ZOG1 polypeptide sequence. Thus, according to one aspect of thedisclosure, nucleic acid molecules that comprise specified regions ofthese sequences are provided. Exemplary of such nucleic acid moleculesare oligonucleotides that are useful as probes or primers to detect andamplify cis-ZOG1-encoding nucleic acids from other plant species. Sucholigonucleotides are useful as hybridization probes or PCR primers, andtypically comprise at least 15 consecutive bases of the disclosedsequences. In other embodiments, such oligonucleotides comprise longerregions of the disclosed sequences, such as at least 20, 25 or 30consecutive nucleotides.

In another aspect, the disclosure provides compositions and methods forisolating nucleic acid sequences having cis-ZOG1 activity from otherplant species. Typically, such methods involve hybridizing probes orprimers derived from the disclosed Zea mays sequences to nucleic acidsobtained or derived from such other plant species.

Homologous and orthologous sequences to the Zea mays cis-ZOG1 nucleicacid and amino acid sequences share key functional and structuralcharacteristics with the disclosed Zea mays sequences. Functionally,such sequences encode (or comprise) a polypeptide that catalyzes theO-glucosylation of cis-zeatin. Structurally, such sequences share aspecified structural relationship with the disclosed sequences. By wayof example, in certain embodiments, homologous amino acid sequences haveat least 70% sequence identity with the Zea mays cis-ZOG1 amino acidsequence. In other embodiments, homologous nucleic acid sequenceshybridize under stringent conditions to the disclosed Zea mays cis-ZOG1nucleic acid sequences.

Another aspect of the disclosure provides purified cis-ZOG1 enzyme.Having provided nucleic acid molecules that encode these enzymes, thedisclosure facilitates the expression of cis-ZOG1 in heterologoussystems, including, but not limited to, E. coli, yeast, and baculovirusexpression systems. Thus, the disclosure permits the large-scaleproduction of the enzymes for agricultural and other applications.

These and other aspects of the disclosure will become readily apparentin light of the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the formation of trans-zeatin-O-glucoside and the formationof cis-zeatin-O-glucoside.

FIG. 2 is a series of graphs showing the analysis of enzymatic reactionproducts by HPLC. FIG. 2A shows the elution profile of the cis-zeatinstandard. FIG. 2B shows the elution profile of products from a reactionwith ¹⁴C-cis-zeatin, recombinant cis-zeatin-O-glucosyltransferase, anduridine diphosphate glucose (UDPG). FIG. 2C shows the elution profile ofreaction products with cis-zeatin after digestion with β-glucosidase.

For FIG. 2B, 200 μL of supernatant obtained from cell culture wasincubated with approximately 1 nmol of labeled zeatin and UDPG for 2hours. For FIG. 2C, Approximately 0.4 nmole of reaction product obtainedfrom reactions similar to FIG. 2B was incubated with β-glucosidase for 4hours to reconvert the product to zeatin.

FIG. 3 shows respective mass spectrum profiles of reaction products withcharacteristic ion of cis-zeatin and the expected molecular mass ofcis-zeatin O-glucoside (FIG. 3A), and standard of trans-zeatinO-glucoside (FIG. 3B).

FIG. 4 shows a comparison of nucleic acid residues 743–1133 of the openreading frame (ORF) from Phaseolus lunatus ZOG1 (SEQ ID NO: 1) andnucleic acid residues 764–1157 of the ORF from Zea mays cis-ZOG1 (SEQ IDNO: 7).

FIG. 5 shows a comparison of amino acid residues 14–459 of the openreading frame (ORF) from Phaseolus lunatus ZOG1 (SEQ ID NO: 2) and aminoacid residues 9–467 of the ORF from Zea mays cis-ZOG1 (SEQ ID NO: 8).

SEQUENCE LISTINGS

The nucleic acid and amino acid sequences listed in the accompanyingsequence listing are shown using standard letter abbreviations fornucleotide bases, and three-letter code for amino acids. Only one strandof each nucleic acid sequence is shown, but the complementary strand isunderstood to be included by any reference to the displayed strand.

SEQ ID NO: 1 shows the nucleic acid sequence of the Phaseolus lunatuszeatin O-glucosyltransferase (ZOG1) open reading frame (ORF).

SEQ ID NO: 2 shows the amino acid sequence of the Phaseolus lunatuszeatin O-glucosyltransferase (ZOG1) open reading frame (ORF).

SEQ ID NO: 3 shows the nucleic acid sequence of Zea mays EST-cdmah36.

SEQ ID NOS: 4 and 5 show the nucleic acid sequences of two polymerasechain reactions (PCR) primers used to amplify corn genomic DNA.

SEQ ID NO: 6 shows the nucleic acid sequence, designated as corn2, ofthe corn DNA fragment amplified by the PCR primers shown in SEQ ID NOS:4 and 5.

SEQ ID NO: 7 shows the nucleic acid sequence of the ORF of cis-zeatinO-glucosyltransferase (cis-ZOG1) from Zea mays.

SEQ ID NO: 8 shows the deduced amino acid sequence of cis-ZOG1.

SEQ ID NOS: 9 and 10 show primers that can be used to amplify the ORFshown in SEQ ID NO: 7 to produce recombinant protein in pTRC 99Aexpression vector.

SEQ ID NO: 11 shows the cDNA sequence encoding cis-ZOG1.

DETAILED DESCRIPTION

I. Abbreviations and Explanations of Terms

A. Abbreviations

¹⁴C-cis-zeatin: cis-[8-¹⁴C]zeatin OXRZ: O-xylosylribosylzeatin cis-Z:cis-zeatin UDPG: uridine diphosphate glucose Z: trans-zeatin UDPX:uridine diphosphate xylose OGZ: O-glucosylzeatin ADPG: adenosinediphosphate glucose OXZ: O-xylosylzeatin TEA: triethylamine DHZ:dihydrozeatin ORF: open reading frame OXDHZ: O-xylosyldihydrozeat- MAb:monoclonal antibody in RZ: ribosylzeatin EST: expressed sequence tagZOG: zeatin O-glucosyltransfer- ase

B. Explanations of Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Lewin, Genes VII, Oxford University Press, 2000 (ISBN0-19-879276-X); Kendrew et al. (eds.), The Encyclopedia of MolecularBiology, Blackwell Science Ltd., 1994 (ISBN 0-632-02182–9); and Meyers(ed.), Molecular Biology and Biotechnology: a Comprehensive DeskReference, VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8). Thenomenclature for DNA bases as set forth at 37 C.F.R § 1.822 and thestandard three-letter codes for amino acid residues are used herein.

In order to facilitate review of the various embodiments, the followingexplanations of terms are provided:

cis-zeatin O-glucosyltransferase (cis-ZOG1): The defining functionalcharacteristic of the cis-ZOG 1 enzyme is its ability to glucosylatecytokinins, such as cis-zeatin, and produce a product that is anO-glucoside of cytokinln. By way of example, the Zea mays cis-ZOG1 iscapable of glucosylating cis-zeatin to form cis-O-glucosylzeatin, aconversion that is depicted in FIG. 1. This is in contrast to thetrans-ZOG 1 isolated from Phaseolus lunatus which glycosylatestrans-zeatin. The cis-ZOG1 activity can be measured using an assaysimilar to that described by Dixon et al. (Dixon et al., Plant Physiol.90:1316–1321, 1989), described in detail below. This disclosure providesa cDNA and a gene encoding the cis-ZOG1 enzyme from Zea mays. However,the disclosure is not limited to this particular cis-ZOG1: othernucleotide sequences that encode cis-ZOG1 enzymes are also encompassedby the disclosure, including variants of the disclosed Zea mays cDNA andorthologous sequences from other plant species, the cloning of which isnow enabled. Such sequences share the essential functionalcharacteristic of encoding an enzyme that is capable of glucosylatingcytokinins. Nucleic acid sequences that encode cis-zeatinO-glucosyltransferases and the proteins encoded by such nucleic acidsshare not only this functional characteristic, but also a specifiedlevel of sequence similarity (or sequence identity), as addressed below.The concept of sequence identity also can be expressed in the ability oftwo sequences to hybridize to each other under stringent conditions.

Isolated: An “isolated” biological component (such as a nucleic acid orprotein or organeue) is a component that has been substantiallyseparated or purified away from other biological components in the cellof the organism in which the component naturally occurs, i.e., otherchromosomal and extra-chromosomal DNA, RNA, proteins, and organelles.Nucleic acids and proteins that have been “isolated” include nucleicacids and proteins purified by standard purification methods. The termalso embraces nucleic acids and proteins prepared by recombinantexpression in a host cell, as well as chemically synthesized nucleicacids.

Operably linked: A first nucleic acid sequence is “operably linked” witha second nucleic acid sequence whenever the first nucleic acid sequenceis placed in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein-coding regions, in the samereading frame.

Ortholog: Two nucleotide or amino acid sequences are orthologs of eachother if they share a common ancestral sequence and diverged when aspecies carrying that ancestral sequence split into two species.Orthologous sequences are also homologous sequences.

Probes and primers: Nucleic acid probes and primers may be readilyprepared based on, the nucleic acid sequences provided. A “probe”comprises an isolated nucleic acid sequence attached to a detectablelabel or reporter molecule. Typical labels include radioactive isotopes,ligands, chemiluminescent agents, and enzymes. Methods for labeling andguidance in the choice of labels appropriate for various purposes arediscussed, e.g., in Sambrook et al. (eds.), Molecular Cloning: ALaboratory Manual, 2nd ed., vol. 1–3, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989; and Ausubel et al. (eds.) CurrentProtocols in Molecular Biology, Greene Publishing andWiley-Interscience, New York (with periodic updates), 1987.

“Primers” are short nucleic acids, preferably DNA oligonucleotides 15nucleotides or more in length, that are annealed to a complementarytarget DNA strand by nucleic acid hybridization to form a hybrid betweenthe primer and the target DNA strand, then extended along the target DNAstrand by a DNA polymerase enzyme. Primer pairs can be used foramplification of a nucleic acid sequence, e.g., by the polymerase chainreaction (PCR) or other nucleic-acid amplification methods known in theart.

As noted, probes and primers are preferably 15 nucleotides or more inlength; to enhance specificity, probes and primers of 20 or morenucleotides may be preferred.

Methods for preparing and using probes and primers are described, forexample, in Sambrook et al. (eds.), Molecular Cloning: A LaboratoryManual, 2nd ed., vol. 1–3, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989; Ausubel et al. (eds.), Current Protocols inMolecular Biology, Greene Publishing and Wiley-Interscience, New York(with periodic updates), 1987; and Innis et al., PCR Protocols: A Guideto Methods and Applications, Academic Press: San Diego, 1990. PCR primerpairs can be derived from a known sequence, for example, by usingcomputer programs intended for that purpose such as Primer™ (Version0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge,Mass.). One of skill in the art will appreciate that the specificity ofa particular probe or primer increases with the length of the probe orprimer. For example, a primer comprising 20 consecutive nucleotides willanneal to a target with a higher specificity than a corresponding primerof only 15 nucleotides. Thus, in order to obtain greater specificity,probes and primers may be selected that comprise, by way of example, 10,20, 25, 30, 35, 40, 50 or more consecutive nucleotides.

Purified: The term “purified” does not require absolute purity; rather,it is intended as a relative term. Thus, for example, a purifiedcis-ZOG1 preparation is one in which the cis-ZOG1 is more enriched thanthe protein in its natural environment within a cell. Preferably, apreparation of cis-ZOG1 is purified such that the cis-ZOG1 represents atleast 50% of the total protein content of the preparation.

Recombinant: A “recombinant” nucleic acid is one having a sequence thatis not naturally occurring or has a sequence made by an artificialcombination of two otherwise-separated, shorter sequences. Thisartificial combination is often accomplished by chemical synthesis or,more commonly, by the artificial manipulation of isolated segments ofnucleic acids, e.g. by genetic engineering techniques.

Sequence identity: The similarity between two nucleic acid sequences orbetween two amino acid sequences is expressed in terms of the level ofsequence identity shared between the sequences. Sequence identity istypically expressed in terms of percentage identity; the higher thepercentage, the more similar the two sequences are.

Methods for aligning sequences for comparison purposes are well known inthe art. Various programs and alignment algorithms are described in:Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J.Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA85:2444, 1988; Higgins & Sharp, Gene 73:237–244, 1988; Higgins & Sharp,CABIOS 5:151–153, 1989; Corpet et al., Nucleic Acids Research16:10881–10890, 1988; Huang, et al., Computer Applications in theBiosciences 8:155–165, 1992; and Pearson et al., Methods in MolecularBiology 24:307–331, 1994. Altschul et al., J. Mol. Biol., 215:403–410,1990, presents a detailed consideration of sequence alignment methodsand homology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST™, Altschul et al. J.Mol. Biol., 215:403–410, 1990) is available from several sources,including the National Center for Biotechnology Information (NCBI,Bethesda, Md.) and on the Internet, for use in connection with thesequence-analysis programs blastp, blastn, blastx, tblastn and tblastx.

For comparisons of amino acid sequences of greater than about 30 aminoacids, the “Blast 2 sequences” function in the BLAST™ program isemployed using the default BLOSUM62 matrix set to default parameters,(gap existence cost of 11, and a per-residue gap cost of 1). Whenaligning short peptides (fewer than about 30 amino acids), the alignmentshould be performed using the Blast 2 sequences function, employing thePAM30 matrix set to default parameters (open gap 9, extension gap 1penalties). Proteins with even greater similarity to the referencesequences will show increasing percentage identities when assessed bythis method, such as at least 45%, at least 50%, at least 60%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least95% sequence identity.

Specific Binding Agent: A “specific binding agent” is an agent that iscapable of specifically binding to the cis-ZOG1, and may includepolyclonal antibodies, monoclonal antibodies (including humanizedmonoclonal antibodies) and fragments of monoclonal antibodies such asFab, F(ab′)2 and Fv fragments, as well as any other agent capable ofspecifically binding to the epitopes on the proteins.

Substantial similarity: A first nucleic acid is “substantially similar”to a second nucleic acid if, when optimally aligned (with appropriatenucleotide insertions or deletions) with the other nucleic acid (or itscomplementary strand), there is nucleotide sequence identity in at leastabout 60%, 75%, 80%, 85%, 90% or 95% of the nucleotide bases. Sequencesimilarity can be determined by comparing the nucleotide sequences oftwo nucleic acids using the BLAST™ sequence analysis software (blastn)available from The National Center for Biotechnology Information. Suchcomparisons may be made using the software set to default settings(expect=10, filter=default, descriptions=500 pairwise, alignments=500,alignment view=standard, gap existence cost=11, per residue existence=1,per residue gap cost=0.85). Similarly, a first polypeptide issubstantially similar to a second polypeptide if they show sequenceidentity of at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% orgreater when optically aligned and compared using BLAST™ software(blastp) using default settings.

Transformed: A “transformed” cell is a cell into which a nucleic acidmolecule has been introduced by molecular biology techniques. As usedherein, the term “transformation” encompasses all techniques by which anucleic acid molecule might be introduced into such a cell, includingtransfection with a viral vector, transformation with a plasmid vector,and introduction of naked DNA by electroporation, lipofection, andparticle gun acceleration.

Transgenic plant: The term “transgenic plant,” as used herein, refers toa plant that contains recombinant genetic material not normally found inplants of this type and that has been introduced into the plant inquestion (or into progenitors of the plant) by human manipulation. Thus,a plant that is grown from a plant cell into which recombinant DNA isintroduced by transformation is a transgenic plant, as are all offspringof the transgenic plant that contain the introduced DNA (whetherproduced sexually or asexually).

Vector: A “vector” is a nucleic acid molecule as introduced into a hostcell, thereby producing a transformed host cell. A vector may includenucleic acid sequences, such as an origin of replication, that permitthe vector to replicate in a host cell. A vector may also include one ormore selectable marker genes and other genetic elements known in theart.

II. Isolation of cDNA and Genomic Sequences Encoding cis-ZeatinO-glucosyltransferase from Zea mays

A. Overview of Experimental Procedures

The sequence of ZOG1 (zeatin O-glucosyltransferase) from Phaseoluslunatus (SEQ ID NO: 1) was compared with sequences contained in an ESTdatabase maintained by Pioneer Hi-Bred International Inc. (PHI).Candidate ESTs were further analyzed. Specific PCR primers were designedto bind to regions of conserved sequences found by comparing theglucosyltransferase, ZOG1, and a selected corn EST, cdmah36. Theseprimers were then used to amplify corn genomic DNA to obtain a probe,CORN2 (SEQ ID NO: 6). The CORN2 probe was then used to screen cDNAlibraries. This screen led to the identification of the cis-ZOG1encoding sequence (SEQ ID NO: 11).

B. Methods

1. Construction of cDNA library from corn kernels. Total RNA wasisolated from 35 DAP Zea mays kernels (inbred B73) using TriPure®reagent following the manufacturer's protocols (Boehringer Mannheim,Germany). mRNA was then isolated using the Poly A Ttract System III,according to the manufacturer's instructions (Promega, Madison, Wis.).The expression/cloning library was constructed using the SuperScript™Plasmid System for cDNA Synthesis and Plasmid Cloning (LifeTechnologies, Inc., Rockville, Md.). First-strand synthesis was primedwith a NotI-oligo-d(T) primer-adapter. Following second-strand synthesisSalI adapters were ligated to the cDNA, which was subsequently digestedwith NotI and size-fractionated. cDNA was directional inserted into apSPORT1™ (Life Technologies, Inc., Rockville, Md.) plasmid following themanufacturer's procedure.

2. Selection of cDNA. The cDNA library was screen with a ³²P-labeledinsert of CORN2 using standard hybridization protocols.

3. Isolation of Recombinant Proteins. To obtain recombinant proteins,the ORF of the selected cDNA was amplified by PCR using primers (SEQ IDNOS: 9 and 10) that generated products having an NcoI site at the 5′ anda XbaI site at the 3′ terminus. The PCR products were digested with NcoIand XbaI restriction enzymes and ligated into a p Trc 99A expressionvector (Pharmacia) modified to contain seven histidine residues (CAT orCAC), and transformed into the XL1 Blue cell line. Colonies wereselected on AMP-LB plates. An individual colony was grown overnight inSOC-AMP media (F. M. Ausubel et al., Short Protocols in MolecularBiology, John Wiley and sons, 1989) and 0.5 mL of the culture was usedto inoculate 50 mL of SOC-AMP media Induction was achieved withisopropyl-D-thiogalactoside (IPTG; 0.5 mL of 0.5 M) after cells wereallowed to grow for 3–4 hours (OD at 595 nm of 0.8–1.0). After 4 hourscells were collected and frozen at −80 C overnight. Cells wereresuspended in 0.5 mL of 0.2 M Tris pH 7.5 containing 1 mg/mL oflysozyme. Samples were incubated on ice for 30 minutes, frozen for 10minutes at −80 C, thawed in cold water, and then DNAase (1 μg/10 μlsupernatant) was added. After 10 minutes at room temperature, thesamples were sonicated with four 15-second bursts to release proteinsfrom cells. Soluble proteins were collected after centrifugation andused for enzyme assays.

4. Enzyme assays and analysis of reaction products. Enzyme activity wasdetermined as reported previously (Dixon et al., Plant Physiol.90:1316–1321, 1989). Briefly, a specified amount of recombinant protein,¹⁴C-labeled cis-zeatin (specific activity of 24 mCi/mmol), and aglycosyl donor (4 mM of UDPG) were incubated in MgCl₂ (0.07 M) in 0.17 MTris, pH 8.0. Reaction products were then analyzed by HPLC (Dixon etal., Plant Physiol. 90:1316–1321, 1989).

5. Isolation of Genomic Sequence of cis-Zeatin O-glucosyltransferase(cis-ZOG1) from Zea mays. Isolation of the genomic sequence was based onthe principles of PCR (Ochman et al., PCR Technology-Principles andApplications for DNA Amplification 105–111, 1989). DNA was isolated fromZea mays using a modified CTAB (hexadecyltrimethylammonium bromide)method (Doyle et al., Focus 12:13–15, 1990). PCR was performed withprimers homologous to the 5′ and 3′ regions of the cDNA clone. To obtaingenomic sequence inclusive of the expressed region (cDNA), standard PCRreactions were performed using pairs of primers based on the sequence ofthe cDNA. The products obtained from PCR were analyzed on a 1% SeaPlaque gel. Bands of interest were excised and DNA was purified withQiaex II™ Gel Extraction Kit (Qiagen, Santa Clarita, Calif.). Theproducts were ligated into a pGem-T vector (Promega, Madison, Wis.) forsequencing.

C. Results

1. Identification of candidate EST, design of PCR primers and synthesisof a specific probe. The sequence of ZOG1 (zeatin O-glucosyltransferase)from Phaseolus lunatus (SEQ ID NO: 1) was compared with sequencescontained in the EST database of Pioneer Hi-Bred International Inc.(PHB). The partial sequence of an EST, cdmah36 (SEQ ID NO: 3) was judgedto have the highest homology. Based on the sequence of ZOG1 andEST-cdmah36, a set of PCR primers was designed (SEQ ID NOS: 4 and 5) toamplify corn genomic DNA to generate a 292-bp probe designated as CORN2(SEQ ID NO: 6).

2. Isolation of the cDNA and the gene. A cDNA library (P0033) from PHBconstructed from 35-DAP (day after pollination) corn kernels of theinbred B73 was probed with CORN2, and a cDNA containing an ORF of 1401bp encoding a protein of 51.1 kD (SEQ ID NOS: 7 and 8, respectively) wasisolated. The corresponding genomic sequence was obtained by PCR usingprimers based on the flanking sequence of the ORF to ampl genomic DNA ofB73. The isolated gene did not contain any introns.

3. Enzyme activity of the gene product. To determine the biologicalactivity and function of the peptide encoded by the selected cDNA, theORF was amplified using the primers (SEQ ID NOS: 9 and 10), digestedwith the restriction enzymes NcoI and XbaI and spliced into the PHTplasmid. Recombinant protein was obtained using protocols previouslydescribed (Martin et al., Proc. Natl. Acad. Sci. USA 96:284–289, 1999a;Martin et al., Plant Physiol. 120:553–557, 1999). The function of therecombinant protein was determined. The protein is a cytokinin metabolicenzyme mediating the formation of O-glucosyl-cis-zeatin from cis-zeatinand UDPG (FIG. 2B). The authenticity of the reaction product (cis-zeatinO-glucoside) was demonstrated by the re-conversion of the reactionproduct to cis-zeatin by β-glucosidase (FIG. 2C), and by the MS profileof the product (FIG. 3), exhibiting the characteristic spectrum ofcis-zeatin but with the expected molecular mass of its O-glucoside. Theenzyme does not use trans-zeatin, trans-ribosylzeatin, dihydrozeatin orribosyldihydrozeatin as the substrate.

The gene is designated as cis-ZOG1 and is deemed new based on BLAST™searches of public databases. The only genes with significant homologyare ZOG1 and ZOX1 of Phaseolus. The enzyme encoded by cis-ZOG1, and thefunction of the enzyme are novel and have not been described previously.

4. Homology to Phaseolus zeatin O-glycosyltransferases ZOG1 and ZOX1.The DNA of cis-ZOG1 is 60% identical to ZOG1 (FIG. 4). However, thelongest contiguous segment is only 14 bp. The amino acid sequence is 41%identical to ZOG1 with a longest identical stretch of amino acids beingten residues (FIG. 5). The amino acid sequence identity to ZOX1 is also41% (not shown because ZOG1 and ZOX1 have 93% and 87% identical DNA andamino acid sequences).

The following examples are illustrative of various embodiments.

EXAMPLE ONE Preferred Method for Producing cis-ZOG1 Nucleic acids

With the provision herein of the cis-ZOG1 ORF, the polymerase chainreaction (PCR) may now be utilized in a preferred method for producingnucleic acid sequences encoding cis-ZOG1 ORF. PCR amplification of thedisclosed Zea mays sequences may be accomplished either by direct PCRfrom a plant cDNA library or by Reverse-Transcription PCR (RT-PCR) usingRNA extracted from plant cells as a template. Methods and conditions forboth direct PCR and RT-PCR are known in the art and are described inInnis et al. (Innis et al., PCR Protocols, A Guide to Methods andApplications, Academic Press Inc., 1990). Suitable plant cDNA librariesfor direct PCR include the Arabidopsis cDNA library described by Newmanet al. (Newman et al., Plant Physiol. 106:1241–1255, 1994) and Zea mayscDNA libraries constructed as described above.

The selection of PCR primers is made according to the portions of thecDNA or gene that are to be amplified. Primers may be chosen to amplifysmall segments of the cDNA, the open reading frame, the entire cDNAmolecule or the entire gene sequence. Variations in amplificationconditions may be required to accommodate primers of differing lengths;such considerations are well known in the art and are discussed in Inniset al. (Innis et al., PCR Protocols, A Guide to Methods andApplications, Academic Press, Inc., 1990), Sambrook et al. (Sambrook etal., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press,1989), and Ausubel et al. (Ausubel et al., Current Protocols inMolecular Biology, Greene Publishing Associates and Wiley-Intersciences,1987).

By way of example only, the entire cis-ZOG1 ORF as shown in SEQ ID NO: 7may be amplified using the following combination of the primers shown inSEQ ID NOS: 9 and 10. These primers are illustrative only; it will beappreciated by one skilled in the art that many different primers may bederived from the provided ORF in order to amplify particular regions ofthis molecule. Resequencing of PCR products obtained by theseamplification procedures is recommended to facilitate confirmation ofthe amplified sequence and also to provide information on naturalvariation on this sequence in different ecotypes and plant populations.

Oligonucleotides that are derived from the disclosed Zea mays sequencesare encompassed within the scope of the present disclosure. Preferably,such oligonucleotide primers will comprise a sequence of at least 15–20consecutive nucleotides of the disclosed Zea mays sequence. To enhanceamplification specificity, oligonucleotide primers comprising at least15, 20, 25, 30, 35, 40, 45 or 50 consecutive nucleotides of thesesequences may also be used.

EXAMPLE TWO Isolation of Homologous Gene Sequence from other PlantSpecies

With the provision herein of the disclosed Zea mays sequence, thecloning of orthologous cDNAs and genes from other plant species bystandard methodologies is now enabled. Thus, the present disclosureincludes methods of isolating both cDNA and genomic sequences encodingcis-ZOG1. Both conventional hybridization and PCR amplificationprocedures may be utilized to clone such sequences. Common to both ofthese techniques is the hybridization of probes or primers derived fromthe disclosed Zea mays sequences to a target nucleotide preparation,which may be, in the case of conventional hybridization approaches, acDNA or genomic library or, in the case of PCR amplification, a cDNA orgenomic library, or an mRNA preparation.

Direct PCR amplification may be performed on cDNA or genomic librariesprepared from the plant species in question, or RT-PCR may be performedusing mRNA extracted from the plant cells using standard methods. PCRprimers will comprise at least 15 consecutive nucleotides of thedisclosed Zea mays sequence. One of skill in the art will appreciatethat sequence differences between the disclosed Zea mays sequence andthe target nucleic acid to be amplified may result in loweramplification efficiencies. To compensate for this, PCR primers from adifferent region of the target sequence may be used. Where lowerannealing temperatures are used, sequential rounds of amplificationusing nested primer pairs may be necessary to enhance specificity.

For conventional hybridization techniques (described further in ExampleFour below) the hybridization probe is preferably conjugated with adetectable label such as a radioactive label, and the probe ispreferably at least 20 nucleotides in length. As is well known in theart, increasing the length of hybridization probes tends to giveenhanced specificity. The labeled probe derived from the Zea mays cDNAor ORF sequence may be hybridized to a plant cDNA or genomic library,and the hybridization signal may be detected using means known in theart. The hybridizing colony or plaque (depending on the type of libraryused) is then purified, and the cloned sequence contained in that colonyor plaque is isolated and characterized.

Homologs of the Zea mays cis-ZOG1 alternatively may be obtained byimmunoscreening of an expression library. With the provision herein ofthe disclosed Zea mays nucleic acid sequence, the enzymes may beexpressed and purified in a heterologous expression system (e.g. E.coli) and used to raise antibodies (monoclonal or polyclonal) specificfor the cis-ZOG1 protein. Antibodies also may be raised againstsynthetic peptides derived from the Zea mays amino acid sequencespresented herein. Methods of raising antibodies are well known in theart and are described in Harlow and Lane (Harlow et al., Antibodies, ALaboratory Manual, Cold Spring Harbor Laboratory, New York, 1988). Suchantibodies then can be used to screen an expression cDNA libraryproduced from the plant from which it is desired to clone the cis-ZOG1gene ortholog, using the methods described above. The selected cDNAs canbe confirmed by sequencing and enzyme activity.

The disclosed Zea mays sequences, and homologs of these sequences fromother plants, may be incorporated into transformation vectors andintroduced into plants to modify cis-ZOG1 activity in such plants, asdescribed in Example Three below. It is anticipated that the nativecis-ZOG1 gene promoter may be useful particularly in the practice of thepresent disclosure in that the promoter may be used to drive theexpression of cis-ZOG1 transgenes, such as antisense constructs. Forexample, by using the native cis-ZOG1 gene promoter, expression of thesetransgenes may be regulated in coordination with the native cis-ZOG1gene (for example, in the same temporal or tissue-specific expressionpatterns).

EXAMPLE THREE Transgenic Plants with Modified cis-ZOG1 Expression

Once a gene (or cDNA) encoding a protein involved in the determinationof a particular plant characteristic has been isolated, standardtechniques may be used to express the cDNA in transgenic plants in orderto modify that particular plant characteristic. The basic approach is toclone the cDNA into a transformation vector, such that the cDNA isoperably linked to control sequences (e.g., a promoter) that directexpression of the cDNA in plant cells. The transformation vector is thenintroduced into plant cells by one of a number of techniques (e.g.,electroporation, Agrobacteria infection and biolistic delivery), andprogeny plants containing the introduced cDNA are selected. Preferablyall or part of the transformation vector will stably integrate into thegenome of the plant cell. That part of the transformation vector thatintegrates into the plant cell and that contains the introduced cDNA andassociated sequences for controlling expression (i.e., the introduced“transgene”) may be referred to as the recombinant expression cassette.

Selection of progeny plants containing the introduced transgene may bemade based upon the detection of an altered phenotype. Such a phenotypemay result directly from the cDNA cloned into the transformation vectoror may be manifested as enhanced resistance to a chemical agent (such asan antibiotic) as a result of the inclusion of a dominant selectablemarker gene incorporated into the transformation vector.

The choice of (a) control sequences and (b) how the cDNA (or selectedportions of the cDNA) are arranged in the transformation vector relativeto the control sequences determine, in part, how the plantcharacteristic affected by the introduced cDNA is modified. For example,the control sequences may be tissue specific, in which instances thecDNA is expressed only in particular tissues of the plant (e.g., pollen,seed) and the affected characteristic is modified only in those tissues.The cDNA sequence may be arranged relative to the control sequence suchthat the cDNA transcript is expressed normally, or in an antisenseorientation. Expression of an antisense RNA corresponding to the clonedcDNA will result in a reduction of the targeted gene product (thetargeted gene product being the protein encoded by the plant gene fromwhich the introduced cDNA was derived). Over-expression of theintroduced cDNA, resulting from a plus-sense orientation of the cDNArelative to the control sequences in the vector, may lead to an increasein the level of the gene product, or may result in co-suppression (alsotermed “sense suppression”) of that gene product.

Successful examples of the modification of plant characteristics bytransformation with cloned cDNA sequences are replete in the technicaland scientific literature. Selected examples, that serve to illustratethe current knowledge in this field of technology, and which are hereinincorporated by reference, include:

U.S. Pat. No. 5,451,514 to Boudet (modification of lignin synthesisusing antisense RNA and co-suppression);

U.S. Pat. No. 5,443,974 to Hitz (modification of saturated andunsaturated fatty acid levels using antisense RNA and co-suppression);

U.S. Pat. No. 5,530,192 to Murase (modification of amino acid and fattyacid composition using antisense RNA);

U.S. Pat. No. 5,455,167 to Voelker (modification of medium chain fattyacids)

U.S. Pat. No. 5,231,020 to Jorgensen (modification of flavenoids usingco-suppression);

U.S. Pat. No. 5,583,021 to Dougherty (modification of virus resistanceby expression of plus-sense untranslatable RNA);

WO 96/13582 (modification of seed VLCFA composition using overexpression, co-suppression and antisense RNA in conjunction with theArabidopsis FAE1 gene); and

WO 95/15387 (modification of seed VLCFA composition using overexpression of jojoba wax synthesis gene).

These examples include descriptions of transformation-vector selection,transformation techniques, and the construction of constructs designedto over-express the introduced cDNA or to express antisense RNAcorresponding to the cDNA. In light of the foregoing and the disclosedZea mays sequences, it is apparent that one of skill in the art will beable to introduce these nucleic acids, or homologous or derivative formsof these molecules (e.g., antisense forms), into plants in order toproduce plants having modified cis-zeatin O-glucosyltransferaseactivity. Modification of the activity of cis-ZOB1 in plants will permitcontrolled modification of not only zeatin function, but also othercytokinins and, as a consequence of the interdependent regulation ofplant hormones, other hormones. The result can be altered plantdevelopment with agricultural and economic consequences.

a. Plant Types

Zeatins are found in all plant types. Thus, DNA molecules (e.g., thecis-ZOG1 cDNA and homologs of this sequence and derivatives such asantisense forms) may be introduced into any plant type in order tomodify the cis-ZOG1 activity in the plant. The sequences of the presentdisclosure may be used to modify cis-zeatin O-glucosyltransferaseactivity in any higher plant, including monocotyledonous,dicotyledenous, and gymnosperm plants, including, but not limited to Zeamays, wheat, rice, barley, soybean, beans in general, rape/canola,alfalfa, flax, sunflower, safflower, brassica, pine trees such asloblolly pine and Douglas fir, cotton, flax, peanut, clover, vegetablessuch as lettuce, tomato, cucurbits, potato, carrot, radish, pea,lentils, cabbage, broccoli, brussel sprouts, peppers; tree fruits suchas apples, pears, peaches, apricots; flowers such as carnations androses.

b. Vector Construction, Choice of Promoters

A number of recombinant vectors suitable for stable transfection ofplant cells or for the establishment of transgenic planes have beendescribed including those described in Pouwels et al., (Pouwels et al.,Cloning Vectors: A Laboratory Manual, 1987), Weissbach and Weissbach,(Weissbach et al., Methods for Plant Molecular Biology, Academic Press,1989), and Gelvin et al., (Gelvin et al., Plant Molecular BiologyManual, Kluwer Academic Publishers, 1990). Typically,plant-transformation vectors include one or more cloned plant genes (orcDNAs) under the transcriptional control of 5′ and 3′ regulatorysequences and a dominant selectable marker. Such plant-transformationvectors typically also contain a promoter-regulatory region (e.g., aregulatory region controlling inducible or constitutive,environmentally-or developmentally-regulated, or cell- ortissue-specific expression), a transcription-initiation start site, aribosome-binding site, an RNA-processing signal, atranscription-termination site, and/or a polyadenylation signal.

Examples of constitutive plant promoters that may be useful forexpressing the cDNA include: the cauliflower mosaic virus (CaMV) 35Spromoter, which confers constitutive, high-level expression in mostplant tissues (see, e.g., Odel et al., Nature 313:810, 1985; Dekeyser etal., Plant Cell 2:591, 1990; Terada at al., Mol. Gen. Genet. 220:389,1990; Benfey et al., Science 250:959–966, 1990); the nopaline synthasepromoter (An et al., Plant Physiol. 88:547, 1988); and the octopinesynthase promoter (Fromm et al., Plant Cell 1:977, 1989).

A variety of plant-gene promoters that are regulated in response toenvironmental, hormonal, chemical, and/or developmental signals, alsocan be used for expression of the cDNA in plant cells. These includepromoters regulated by: (a) heat (Callis et al., Plant Physiol. 88:965,1988; Ainley et al., Plant Mot. Biol. 22:13–23, 1993; Gilmartin et al.,The Plant Cell 4:839–949, 1992); (b) light (e.g., the pea rbcS-3Apromoter, Kuhlemeier et al., Plant Cell 1:471, 1989, and the Zea maysrbcS promoter, Schaffner et al., Plant Cell 3:997, 1991); (c) hormones,such as abscisic acid (Marcotte et al., Plant Cell 1:969, 1989); (d)wounding (e.g., wunI, Siebertz et al., Plant Cell 1:961, 1989); and (e)chemicals such as methyl jasminate or salicylic acid (see also Gatz etal., Ann Rev. Plant Physiol. Plant Mol. Biol. 48:89–108, 1997) also canbe used to regulate gene expression.

Alternatively, tissue specific (root, leaf, flower, and seed, forexample) promoters (Carpenter et al., The Plant Cell 4:557–571, 1992;Denis et al., Plant Physiol. 101:1295–1304, 1993; Opperman et al.,Science 263:221–223, 1993; Stockhause et al., The Plant Cell 9:479–489,1997; Roshal et al., EMBO J. 6:1155, 1987; Schernthaner et al., EMBO J.7:1249, 1988; and Bustos et al., Plant Cell 1:839, 1989) can be fused tothe coding sequence to obtain particular expression in respectiveorgans. In addition, the timing of the expression can be controlled byusing promoters such as those acting at senescence (Gan et al., Science270:1936–1988, 1995) or late seed development (Odell et al., PlantPhysiol. 106:447–458, 1994).

Plant-transformation vectors also may include RNA processing signals,for example, introns, that may be positioned upstream or downstream ofthe ORF sequence in the transgene. In addition, the expression vectorsalso may include additional regulatory sequences from the3′-untranslated region of plant genes, e.g., a 3′-terminator region toincrease mRNA stability of the mRNA, such as the PI-II terminator regionof potato or the octopine or nopaline synthase 3′-terminator regions.

Finally, as noted above, plant-transformation vectors also may includedominant selectable marker genes to allow for the ready selection oftransformants. Such genes include those encoding antibiotic-resistancegenes (e.g., resistance to hygromycin, kanamycin, bleomycin, G418,streptomycin, or spectinomycin) and herbicide-resistance genes (e.g.phosphinothricin acetyltransferase).

c. Arrangement of cis-ZOG1 Sequence in Vector

The particular arrangement of the cis-zeatin O-glucosyltransferasesequence in the transformation vector will be selected according to thetype of expression of the sequence that is desired.

Where enhanced cis-zeatin O-glucosyltransferase activity is desired inthe plant, the cis-zeatin O-glucosyltransferase can be ligated to aconstitutive high-level promoter such as the CaMV 35S promoter. As notedbelow, modification of cis-zeatin O-glucosyltransferase synthesis alsomay be achieved by introducing into a plant a transformation vectorcontaining a variant form of the disclosed Zea mays sequences. Forexample, a form can vary from the exact nucleotide sequence of thecis-zeatin O-glucosyltransferase ORF (SEQ ID NO: 7), but still encode aprotein that retains the functional characteristic of the cis-zeatinO-glucosyltransferase protein, i.e., O-glucosylation of cytokinins, suchas cis-zeatin.

In contrast, a reduction of cis-zeatin O-glucosyltransferase activity inthe transgenic plant may be obtained in a number of different ways. Forexample, a reduction in protein product can be achieved through the useof antisense sequences, ribozymes, co-suppression, untranslatable RNAs,and/or dominant negative mutants.

For antisense suppression, the disclosed Zea mays sequences are arrangedin reverse orientation relative to the promoter sequence in thetransformation vector. The introduced sequence need not be thefull-length version of the disclosed Zea mays sequence, and need not beexactly homologous to the endogenous cis-zeatin O-glucosyltransferasefound in the plant type to be transformed. Generally, however, where theintroduced sequence is of shorter length, a higher degree of homology tothe endogenous zeatin O-glucosyltransferase sequence will be needed foreffective antisense suppression. Preferably, the introduced antisensesequence in the vector will be at least 30 nucleotides in length, andimproved antisense suppression will typically be observed as the lengthof the antisense sequence increases. Preferably, the length of theantisense sequence in the vector will be greater than 100 nucleotides.Transcription of an antisense construct as described results in theproduction of RNA molecules that are the reverse complement of mRNAmolecules transcribed from the endogenous cis-zeatinO-glucosyltransferase gene in the plant cell. Although the exactmechanism by which antisense RNA molecules interfere with geneexpression has not been elucidated, it is believed that antisense RNAmolecules bind to the endogenous mRNA molecules and thereby inhibittranslation of the endogenous mRNA.

Suppression of endogenous cis-ZOG1 expression also can be achieved usingribozymes. Ribozymes are synthetic RNA molecules that possess highlyspecific endoribonuclease activity. The production and use of ribozymesare disclosed in U.S. Pat. No. 4,987,071 to Cech and U.S. Pat. No.5,543,508 to Haselhoff. The inclusion of ribozyme sequences withinantisense RNAs may be used to confer RNA-cleaving activity on theantisense RNA, such that endogenous mRNA molecules that bind to theantisense RNA are cleaved, which in turn leads to an enhanced antisenseinhibition of endogenous gene expression.

Constructs in which the disclosed Zea mays sequences (or variantsthereof) are overexpressed may also be used to obtain co-suppression ofthe endogenous cis-ZOG1 in the manner described in U.S. Pat. No.5,231,021 to Jorgensen. Such co-suppression (also termed “sensesuppression”) does not require that the entire disclosed Zea mayssequence be introduced into the plant cells, nor does it require thatthe introduced sequence be exactly identical to the endogenous cis-ZOG1sequence. However, as with antisense suppression, the suppressiveefficiency will be enhanced as: (1) the introduced sequence islengthened, and (2) the sequence similarity between the introducedsequence and the endogenous cis-ZOB1 gene is increased.

Constructs expressing an untranslatable form of the cis-ZOG1 mRNA alsomay be used to suppress the expression of endogenous cis-ZOG1. Methodsfor producing such constructs are described in U.S. Pat. No. 5,583,021to Dougherty et al. Preferably, such constructs are made by introducinga premature stop codon into the cis-ZOG1 ORF.

Finally, dominant negative mutant forms of the disclosed Zea maysproteins may be used to block endogenous cis-ZOG1 activity. Such mutantsrequire the production of mutated forms of the cis-ZOG1 protein thatbind to zeatin but do not catalyze the enzymatic step.

d. Transformation and Regeneration Techniques

Transformation and regeneration of both monocotyledonous anddicotyledonous plant cells are now routine, and the selection of themost appropriate transformation technique will be determined by thepractitioner. The choice of method will vary with the type of plant tobe transformed; those skilled in the art will recognize the suitabilityof particular methods for given plant types. Suitable methods mayinclude, but are not limited to: electroporation of plant protoplasts;liposome-mediated transformation; transformation mediated bypolyethylene glycol (PEG); transformation using viruses; micro-injectionof plant cells; micro-projectile bombardment of plant cells; vacuuminfiltration; and transformation mediated by Agiobacterium tumefaciens(AT). Typical procedures for transforming and regenerating plants aredescribed in the patent documents listed at the beginning of thissection.

e. Selection of Transformed Plants

Following transformation and regeneration of plants with thetransformation vector, transformed plants are preferably selected usinga dominant selectable marker incorporated into the transformationvector. Typically, such a marker will confer antibiotic resistance onthe seedlings of transformed plants, and selection of transformants canbe accomplished by exposing the seedlings to appropriate concentrationsof the antibiotic.

After transformed plants are selected and grown to maturity, they can beassayed using the methods described herein to determine whether cis-ZOG1activity has been altered as a result of the introduced transgene. Inaddition, antisense or sense suppression of the endogenous cis-ZOG1 maybe detected by analyzing mRNA expression on Northern blots.

EXAMPLE FOUR Production of Sequence Variants

As noted above, modification of cis-zeatin O-glucosyltransferaseactivity in plant cells can be achieved by transforming plants with thedisclosed Zea mays sequences, antisense constructs based on thedisclosed Zea mays sequences, or other variants of the disclosed Zeamays sequences. With the provision of the disclosed Zea mays sequencesherein, the creation of variants on these sequences by standardmutagenesis techniques is now enabled.

Variant DNA molecules include those created by standard DNA mutagenesistechniques, for example, M13 primer mutagenesis. Details of thesetechniques are provided in Ch. 15 of Sambrook et al. (Sambrook et al.,Molecular Cloning. A Laboratory Manual, Spring Harbor, N.Y., 1989). Bythe use of such techniques, variants may be created that differ in minorways from the disclosed Zea mays sequences. DNA molecules and nucleotidesequences that are derivatives of those specifically disclosed hereinand that differ from those disclosed by the deletion, addition, orsubstitution of nucleotides while still encoding a protein thatpossesses the functional characteristic of the cis-zeatinO-glucosyltransferase protein (i.e., the ability to convert cis-zeatinto cis-O-glucosylzeatin) are comprehended by this disclosure. DNAmolecules and nucleotide sequences that are derived from the disclosedZea mays sequences include DNA sequences that hybridize under stringentconditions to the DNA sequences disclosed, or fragments thereof.

Hybridization conditions resulting in particular degrees of stringencywill vary depending upon the nature of the hybridization method ofchoice and the composition and length of the hybridizing DNA used.Generally, the temperature of hybridization and the ionic strength(especially the Na⁺ concentration) of the hybridization buffer willdetermine the stringency of hybridization. Calculations regardinghybridization conditions required for attaining particular degrees ofstringency are discussed by Sambrook et al. (Sambrook et al., MolecularCloning: A Laboratory Manual, Spring Harbor, N.Y., 1989), chapters 9 and11, herein incorporated by reference. By way of illustration only, ahybridization experiment may be performed by hybridization of a DNAmolecule (for example, a variant of the Zea mays cis-zeatinO-glucosyltransferase ORF sequence) to a target DNA molecule (forexample, the Zea mays cis-zeatin O-glucosyltransferase gene sequence)which has been electrophoresed in an agarose gel and transferred to anitrocellulose membrane by Southern blotting (Southern, J. Mol. Biol.98:503, 1975). This technique is well known in the art and described inSambrook et al., Molecular Cloning: A Laboratory Manual, Spring Harbor,N.Y., 1989. Hybridization with a target probe labeled with [³²P]-dCTP isgenerally carried out in a solution of high ionic strength such as 6×SSCat a temperature that is 20–25° C. below the melting temperature, T_(m),described below. For such Southern hybridization experiments where thetarget DNA molecule on the Southern blot contains 10 ng of DNA or more,hybridization typically is carried out for 6–8 hours using 1–2 ng/mLradiolabeled probe (of specific activity equal to 10⁹ CPM/μg orgreater). Following hybridization, the nitrocellulose filter is washedto remove background hybridization. The washing conditions should be asstringent as possible to remove background hybridization while retaininga specific hybridization signal. The term T_(m) represents thetemperature above which, under the prevailing ionic conditions, theradiolabeled probe molecule will not hybridize to its target DNAmolecule. The T_(m) of such a hybrid molecule may be estimated from thefollowing equation (Bolton et al., Proc. Natl. Acad. Sci. USA 48:1390,1962):T _(m)=81.5_(E) C−16.6(log ₁₀[Na⁺])+0.41(% G+C)−0.63(%formamide)−(600/l)Wherein l=the length of the hybrid in base pairs. This equation is validfor concentrations of Na⁺ in the range of 0.01 M to 0.4 M, but it isless accurate for calculations of T_(m) in solutions of higher [Na⁺].The equation is also primarily valid for DNAs whose G+C content is inthe range of 30% to 75%, and it applies to hybrids greater than 100nucleotides in length (the behavior of oligonucleotide probes isdescribed in detail in Ch. 11 of Sambrook et al., Molecular Cloning: ALaboratory Manual, Spring Harbor, N.Y., 1989).

Thus, by way of example, for a 150 base pair DNA probe derived from thefirst 150 base pairs of the open reading frame of the Zea mays cis-ZOG1cDNA (with a hypothetical % GC=45%), a calculation of hybridizationconditions required to give particular stringencies may be made asfollows:

For this example, it is assumed that the filter will be washed in0.3×SSC solution following hybridization, thereby [Na⁺]=0.045M; %GC=45%; formamide concentration=0; l=150 base pairs; and T_(m)=81.5°C.−16(log ₁₀[Na⁺])+(0.41×45)−(600/150)=74.4° C.

The T_(m) of double-stranded DNA decreases by 1–1.5° C. with every 1%decrease in homology (Bonner et al., J. Mol. Biol. 81:123, 1973).Therefore, for this example, washing the filter in 0.3×SSC at 59.4–64.4°C. will produce a stringency of hybridization equivalent to 90%.Alternatively, washing the hybridized filter in 0.3×SSC at a temperatureof 65.4–68.4° C. will yield a hybridization stringency of 94%. The aboveexample is provided entirely by way of theoretical illustration. Oneskilled in the art will appreciate that other hybridization techniquesmay be utilized, and that variations in experimental conditions willnecessitate alternative calculations for stringency.

DNA sequences from plants that encode a protein having cis-zeatinO-glucosyltransferase activity and that hybridize under hybridizationconditions of at least 75%, more preferably at least 80%, morepreferably at least 85%, more preferably at least 90% and mostpreferably at least 95% stringency are encompassed within the presentdisclosure.

The degeneracy of the genetic code further widens the scope of thepresent disclosure as the degeneracy enables major variations in thenucleotide sequence of a DNA molecule while maintaining the amino acidsequence of the encoded protein. For example, the second amino acidresidue of the Zea mays cis-ZOG1 protein is alanine. This is encoded inthe Zea mays cis-ZOG1 open reading frame by the nucleotide codon tripletGCG. Because of the degeneracy of the genetic code, three othernucleotide codon triplets-GCA, GCC and GCT-also code for alanine. Thus,the nucleotide sequence of the Zea mays cis-ZOG1 ORF could be changed atthis position to any of these three codons without affecting the aminoacid composition of the encoded protein or the characteristics of theprotein. Based upon the degeneracy of the genetic code, variant DNAmolecules may be derived from the cDNA and gene sequences disclosedherein using standard DNA mutagenesis techniques as described above, orby synthesis of DNA sequences. Thus, this disclosure also encompassesnucleic acid sequences which encode a cis-ZOG1 protein but which varyfrom the disclosed nucleic acid sequences by virtue of the degeneracy ofthe genetic code.

One skilled in the art will recognize that DNA-mutagenesis techniquesmay be used not only to produce variant DNA molecules, but alsofacilitate the production of proteins that differ in certain structuralaspects from the disclosed Zea mays proteins, such proteins beingclearly derivative of the disclosed Zea mays proteins and retaining theessential functional characteristics of cis-ZOG1. Newly derived proteinsalso may be selected in order to obtain variations on thecharacteristics of the disclosed Zea mays proteins, as will be morefully described below. Such derivatives include those with variations inamino acid sequence including minor deletions, additions andsubstitutions, and protein fusions that contain a catalyticallyfunctional region of a cis-ZOG1 protein or sequence variant or orthologthereof.

While the site for introducing an amino acid sequence variation ispredetermined, the mutation per se need not be predetermined. Forexample, in order to optimize the performance of a mutation at a givensite, random mutagenesis may be conducted at the target codon or regionand the expressed protein variants screened for the optimal combinationof desired activity. Techniques for making substitution mutations atpredetermined sites in DNA having a known sequence as described aboveare well known.

Amino acid substitutions are typically of single residues. Within asingle substituted amino acid sequence, there may occur multiplesubstitutions, for instance two or more, 5 or more, 10 or more, 15 ormore, 30 or more, or as many as 50 substitutions within a singlepolypeptide. In some embodiments, the number of amino acid substitutionsis measured based on a percentage of the overall length of the resultantpolypeptide, and thus it is contemplated that there may be about 0.5%,about 1%, about 3%, about 5%, about 7%, about 10%, about 15%, or moresubstitutions within a single polypeptide.

Insertions usually will be on the order of about from 1 to 10 amino acidresidues; and deletions will range about from 1 to 30 residues. In someembodiments, longer insertions (such as fusions) and/or deletions arecontemplated. Deletions or insertions may be made in adjacent pairs,i.e., a deletion of 2 residues or insertion of 2 residues, or multiplesof pairs. Substitutions, deletions, insertions, or any combinationthereof may be combined to arrive at a final construct. Obviously, themutations that are made in the DNA encoding the protein should not placethe sequence out of reading frame and usually will not createcomplementary regions that could produce secondary mRNA structure.

Also contemplated herein are variants of the provided proteins, whichare fusion proteins. Fusion proteins contain relatively long lengths ofadditional amino acids, generally of greater than 10 amino acids inlength, for instance at least 50, at least 100, at least 150, at least200, at least 250, at least 500, at least 750, at least 1000, or moreamino acids.

Substitutional variants are those in which at least one residue in theamino acid sequence has been removed and a different residue inserted inits place. Such substitutions generally are made in accordance with thefollowing Table 1 when it is desired to finely modulate thecharacteristics of the protein. Table 1 shows amino acids that may besubstituted for an original amino acid in a protein and that areregarded as conservative substitutions.

TABLE 1 Listing of Conservative Amino Acid Substitutions OriginalConservative Residue Substitutions ala ser arg lys asn gln; his asp glucys ser gln asn glu asp gly pro his asn; gln ile leu, val leu ile; vallys arg; gln; glu met leu; ile phe met; leu; tyr ser thr thr ser trp tyrtyr trp; phe val ile; leu

Substantial changes in enzymatic function or other features are made byselecting substitutions that are less conservative than those in Table1, i.e., selecting residues that differ more significantly in theireffect on maintaining: (a) the structure of the polypeptide backbone inthe area of the substitution, for example, as a sheet or helicalconformation, (b) the charge or hydrophobicity of the molecule at thetarget site, or (c) the bulk of the side chain. The substitutions thatin general are expected to produce the greatest changes in proteinproperties will be those in which: (a) a hydrophilic residue, e.g. serylor threonyl, is substituted for (or by) a hydrophobic residue, e.g.,leucyl, isoleucyl, phenylalanyl, valyl, or alanyl; (b) a cysteine orproline is substituted for (or by) any other residue; (c) a residuehaving an electropositive side chain, e.g. lysyl, arginyl, or histadyl,is substituted for (or by) an electronegative residue, e.g., glutamyl oraspartyl; or (d) a residue having a bulky side chain, e.g.,phenylalanine, is substituted for (or by) one not having a side chain,e.g., glycine.

The effects of these amino acid substitutions or deletions or additionsmay be assessed for derivatives of the cis-ZOG1 protein by analyzing theability of the derivative proteins to catalyze the conversion ofcis-zeatin to cis-O-glucosylzeatin. These assays may conveniently beperformed using the assay described above.

EXAMPLE FIVE Production of Recombinant cis-ZOG1 Using HeterologousExpression Systems

Many different expression systems are available for expressing clonednucleic acid molecules. Examples of prokaryotic and eukaryoticexpression systems that are routinely used in laboratories are describedin Chapters 16–17 of Sambrook et al. (Sambrook et al., MolecularCloning: A Laboratory Mamual, Spring Harbor, N.Y., 1989), incorporatedherein by reference. Such systems may be used to express cis-ZOG1 athigh levels to facilitate purification of the enzyme. The enzyme may beused for a variety of purposes. For example, the enzyme may be applieddirectly to plants to modulate zeatin function. Alternatively thepurified enzyme produced by recombinant means may be used to synthesizeother zeatin metabolites in vitro, particularly radio- orfluorescent-labeled forms of cis-O-glucosylzeatin. These molecules maybe used as tracers to determine the location in plant tissues and cellsof zeatin and its metabolites. In addition, the recombinant form of theenzyme may be used to produce labeled forms of metabolites of othersubstrates (for example, isoprenylated proteins) on which it may act.The purified recombinant enzyme may also be used as an immunogen toraise enzyme-specific antibodies. Such antibodies are useful as researchreagents (such as in the study of cytokinin regulation in plants) andcan be used diagnostically to determine expression levels of the enzymein agricultural products, including seed.

By way of example only, high-level expression of the cis-ZOG1-proteinmay be achieved by cloning and expressing the cDNA in yeast cells usingthe pYES2 yeast-expression vector (InVitrogen, San Diego, Calif.). Therecombinant cis-ZOG1 may be supplied in the harvested yeast cells (forsubsequent processing). Alternatively, a genetic construct may beproduced to direct secretion of the recombinant cis-ZOG1 from the yeastcells into the medium. This approach will facilitate the purification ofthe cis-ZOG1, if necessary. Secretion of the recombinant cis-ZOG1 fromthe yeast cells may be achieved by placing a yeast-signal sequenceadjacent to the cis-ZOG1 coding region. A number of yeast-signalsequences have been characterized, including the signal sequence foryeast invertase. This sequence has been successfully used to direct thesecretion of heterologous proteins from yeast cells, including proteinssuch as human interferon (Chang et al., Mol. and Cell. Biol.6:1812–1819, 1986), human lactoferrin (Liang et al., J. Agric. FoodChem. 41:1800–1807, 1993), and prochymosin (Smith et al., Science229:1219–1224, 1985).

Alternatively, the enzyme may be expressed at high level in prokaryoticexpression systems, such as E. coli as described above.

Having illustrated and described the principles of the disclosure inmultiple embodiments and examples, it should be apparent to thoseskilled in the art that the provided compositions and methods can bemodified in arrangement and detail without departing from suchprinciples. We claim all modifications coming within the spirit andscope of the following claims.

1. An isolated nucleic acid molecule, comprising: a nucleic acidsequence which encodes a polypeptide comprising the amino acid sequenceof SEQ ID NO: 8, wherein the nucleic acid molecule encodes a polypeptidethat catalyzes the conversion of cis-zeatin to cis-O-glucosylzeatin. 2.The isolated nucleic acid molecule of claim 1, wherein the nucleic acidsequence comprises the nucleic acid sequence set forth in SEQ ID NO: 7,SEQ ID NO: 11, or a nucleic acid sequence that differs from SEQ ID NO: 7or SEQ ID NO: 11 by virtue of the degeneracy of the genetic code withoutaffecting the amino acid composition of the encoded protein.
 3. Theisolated nucleic acid molecule of claim 2, wherein the nucleic acidsequence is the nucleic acid sequence as set forth in SEQ ID NO: 7 orSEQ ID NO:
 11. 4. A recombinant nucleic acid molecule, comprising apromoter sequence operably linked to the nucleic acid molecule ofclaim
 1. 5. A transgenic plant, comprising the recombinant nucleic acidmolecule of claim 4.